Circulating phosphate is a critical determinant of growth plate maturation. Hypophosphatemia impairs apoptosis of hypertrophic chondrocytes in vivo and in vitro, leading to the development of rickets in growing animals. Phosphate induces Erk1/2 phosphorylation and activates the mitochondrial apoptotic pathway in hypertrophic but not proliferative chondrocytes. Inhibition of Erk1/2 phosphorylation in vitro and in vivo impairs phosphate-mediated hypertrophic chondrocyte apoptosis. Studies in Specific Aim I will identify the factors involved in phosphate-mediated apoptosis of hypertrophic chondrocytes upstream to and downstream of Erk1/2 phosphorylation. They will examine the role of C-Raf in Erk1/2 activation in vitro and in vivo and examine the effect of phosphate on activation of, and subcellular localization of, proapoptotic factors. Investigations in Specific Aim II will demonstrate the critical role of C-Raf in growth plate maturation and hypertrophic chondrocyte apoptosis in mice with chondrocyte-specific C-Raf ablation and in primary chondrocytes isolated from these mice. Unlike other hypophosphatemic mouse models, Npt2a null mice have high levels of 1,25-dihydroxyvitamin D and had not been reported to develop rickets. Our studies revealed a rachitic phenotype in these mice at 16 days of age, which resolves by 35 days. Impairing 1,25-dihydroxyvitamin D action in Npt2a null mice leads to progressive rickets, demonstrating that 1,25-dihydroxyvitamin D can compensate for low phosphate in maintaining a normal growth plate. Evidence from our studies implicate enhanced PTH/PTHrP signaling in the etiology of rickets, thus we propose to treat Hyp mice from 2 days of age, with chronic repletion versus high dose intermittent 1,25-dihydroxyvitamin D to determine whether these "physiological" or "pharmacological" interventions normalize the growth plates of the Hyp mice. Parathyroid function and PTHrP expression will also be examined to dissect the molecular basis for the response to these two dosing regimens. The effects of 1,25-dihydroxyvitamin D on the growth plate and on parathyroid function will be compared to that of direct inhibition of FGF23 action using blocking antibodies. The effects of these three treatments on bone will be evaluated histologically, histomorphometrically and by microCT analyses. Thus, the investigations in this proposal are directed at addressing the pathophysiologic basis for the abnormalities observed in XLH and at examining the comparative efficacy of therapeutic interventions, using the Hyp mouse as a model. PUBLIC HEALTH RELEVANCE: Normal skeletal development and maturation requires optimal mineral ion levels. While vitamin D deficiency is once again emerging as a cause of impaired growth and skeletal mineralization in western countries, much of this phenotype is a direct consequence of low levels of circulating mineral ions. Studies in cells and in intact animals, including the mouse model of the human disorder X-linked hypophosphatemia, are expected to elucidate the molecular basis for the growth plate changes associated with low circulating phosphate. The characterization of the therapeutic interventions proposed, on the growth plate, as well as on bone, will help design new therapeutic approaches to the treatment of this disorder, as well as other conditions associated with excessive renal phosphate losses.